A Proteomic Study of Clavibacter Michiganensis Subsp. Michiganensis Culture Supernatants

Abstract

Clavibacter michiganensis, subsp. michiganensis is a Gram-positive plant pathogen infecting tomato (Solanum lycopersicum). Despite a considerable economic importance due to significant losses of infected plants and fruits, knowledge about virulence factors of C. michiganensis subsp. michiganensis and host-pathogen interactions on a molecular level are rather limited. In the study presented here, the proteome of culture supernatants from C. michiganensis subsp. michiganensis NCPPB382 was analyzed. In total, 1872 proteins were identified in M9 and 1766 proteins in xylem mimicking medium. Filtration of supernatants before protein precipitation reduced these to 1276 proteins in M9 and 976 proteins in the xylem mimicking medium culture filtrate. The results obtained indicate that C. michiganensis subsp. michiganensis reacts to a sucrose- and glucose-depleted medium similar to the xylem sap by utilizing amino acids and host cell polymers as well as their degradation products, mainly peptides, amino acids and various C5 and C6 sugars. Interestingly, the bacterium expresses the previously described virulence factors Pat-1 and CelA not exclusively after host cell contact in planta but already in M9 minimal and xylem mimicking medium.

Surface and Extracellular Proteome of the Emerging Pathogen Corynebacterium ulcerans

“…For preparation of extracellular proteins, the cells were separated by centrifugation (25 min, 5000× g , 4 °C) and Complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany) was added to the supernatant to avoid proteolysis. To prevent contamination of extracellular proteins by cells or cell debris, the supernatant was centrifuged again (1 h, 7000× g , 4 °C) and subsequently filtered using 0.2 µm pore-size filters (Minisart, Sartorius, Göttingen, Germany) [ 27 ]. Proteins were precipitated under constant stirring by dropwise addition of trichloroacetic acid (10% w / v ) and incubation at 4 °C overnight.…”

“…Mass spectrometric analyses by nanoLC-MS/MS were carried out as described [ 27 ]. In short, resulting peptides (approx.…”

“…1 µg) were loaded on a nanoflow Ultimate 3000 HPLC (Dionex, Sunnyvale, CA, USA) for separation on EASY-Spray column (Thermo Fisher Scientific; C18 with 2 μm particle size, 50 cm × 75 μm), with a flow rate of 200 nL/min by increasing acetonitrile concentrations over 120 min. All samples were analyzed on an Orbitrap Fusion (Thermo Fisher Scientific, Waltham, MA, USA) with the previously described MS/MS settings [ 27 ]. The mass spectrometer was operating with 2000 V spray voltage, 300–2000 ( m / z ) scan range, a maximum injection time of 50 ms, and an AGC target of 400,000 for the first stage of mass analysis t (MS 1 ).…”

Proteome Profile and Genome Refinement of the Tomato‐Pathogenic Bacterium Clavibacter michiganensis subsp. michiganensis

“…Two plasmids, pCM1 and pCM2, were also identified in Cmm 382 and are necessary for full virulence . Subsequent omics analyses of Cmm have validated the annotated genome and provided evidence for expression of putative virulence genes . A recent comparative genomic study of 16 pathogenic Cmm isolates identified 13 core Sec‐dependent secreted proteins .…”

Cellular and Extracellular Proteome of the Animal Pathogen Corynebacterium silvaticum, a Close Relative of Zoonotic Corynebacterium ulcerans and Corynebacterium pseudotuberculosis

“…Since we are interested in the analysis of pathogenicity determinants of corynebacteria [ 24 , 25 , 26 , 27 ] and different aspects of bacterial proteomics [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ], we started a characterization of the cellular and extracellular proteome of C. silvaticum isolate W25, which is presented here. In total, 1305 proteins were identified out of 2013 proteins encoded by the W25 genome sequence including a number of putative virulence factors such as phospholipase D and sialidase.…”